Antibodies are composed of two chains termed light and heavy chains. The light chain contains an amino-terminal variable domain (VL domain) and a carboxy-terminal constant domain (CL). The heavy chain is composed of an amino-terminal variable domain (VH) and three constant domains (CH1, CH2, CH3). The antibody binding site is located in the VL and VH domains and is made up by six hypervariable loops that represent the Complementarity-Determining Regions (CDRs). Both VL and VH regions contain three CDR loops (CDR1, CDR2 and CDR3), which are connected to a structurally conserved beta.-sheet framework.
With the development of hybridoma technology, it became possible to produce a single population of antibodies, or monoclonal antibodies (mAbs), that specifically targeted a single epitope opening a revolution in the drug discovery field. However, problems with antibody production and in vivo responses, including immunogenicity and cytokine-associated side-effects, have lead to the investigation of altering antibody structure and/or function while still retaining immunospecific binding. Studies have attempted to reduce the antibody to its smallest functional form, without significantly changing the antigen recognition and affinity. The identification of the smallest antibody fragment that is capable of binding to antigen has evolved from full antibody molecules or IgG to Fab and recombinant single chain Fv fragments.
Due to the progress in gene recombination in the 80's, the rapid and easy generation of recombinant variable domains was possible. Using the polymerase chain reaction, diverse repertoires of genes encoding VH and VL domains were cloned from the genomic DNA of immunized animals, allowing the characterization of multiple binding activities and functionalities against several antigens. Nevertheless, the variable domain fragments initially isolated were scarcely soluble and difficult to produce.
Problems in production were addressed with the characterization of camelid antibodies, which are dimeric molecules that comprise only heavy chains. Not only did the discovery of the dimeric molecule address many issues with respect to recombinant antibody production, but the molecule also highlighted the possibility that the heavy chain of immunoglobulin molecules could direct immunospecific binding in the absence of a light chain. It is now widely accepted that Ig heavy chains may retain significant antigen binding ability in the absence of a light chain. There is also evidence from structural studies that the CDR3 region of the VH domain is the most significant of the CDR domains with respect to immunospecificity. This is based on the findings that the HCDR3 amino acid residues provide most of the surface contact area and are crucial in the molecular interaction with the antigen. Accordingly, further reduction of antigen binding protein size to single domain binding proteins may be possible based upon immunoglobulin VH domains.